Use of markers to identify the transformed cells/morphogenic units is an essential component of transformation technology (Puchta 2003). Selection of transformed cells, which are generally few in number and are mingled with a large number of untransformed cells, is possible only through use of selective agents, which can rapidly inhibit growth and/or kill the non-transformed cells. This has been traditionally accomplished through the introduction of antibiotic or herbicide resistance genes, enabling the selection of transgenic cells on media supplemented with the corresponding antibiotic/herbicide (Brasileiro and Dusi 1999; Puchta 2003).
Antibiotics are well documented to severely curtail cellular metabolism in prokaryotes and also affect plant cells by interfering with essential metabolic events in mitochondria and plastids, in particular chloroplasts (Nap et al. 1992; Brasileiro and Aragao 2001; Day 2003). These double membrane organelles involved in energy transduction have been well documented to play a vital role in cellular metabolism and regulation of morphogenesis/developmental events in plant cells. Antibiotics like neomycin are used in medicine for killing pathogenic bacteria such as Staphylococci (Hendley and Ashe 2003). The antibiotic resistance marker genes have potency to make antibiotics ineffective, usually, by phosphorylation (Brasileiro and Aragão 2001; Wright 2005). Similarly, herbicide resistance marker genes such as aroAlepsps and bar genes, used as markers to select the trans-formed cells have the potential to detoxify herbicides such as glyphosate (Comai et al., 1983, 1985; Shah et al. 1986; Clemente et al. 2000) and phosphinothricin (De Block et al. 1987; Barcelo et al. 1994), respectively.
The antibiotic/herbicide resistance marker genes are widely classified in the category of negative selection markers (Day 2003; He et al. 2004). Earlier investigations involved raising choline oxidase transgenic genotypes of Brassica juncea cv. Varuna exploiting one such antibiotic resistance marker, neomycin phosphotransferase II (npt II). Presence of npt II in transformed cells/morphogenic units enables them to resist exogenous kanamycin as product of this gene, neomycin phosphotransferase phosphorylates active kanamycin to an inactive/ineffective form (Bevan et al. 1983; Herrera-Estrella et al. 1983; Brasileiro and Aragão 2001).
However, escape of antibiotic resistance markers like npt II into pathogenic bacteria can make them resistant to the antibiotic, rendering the use of the antibiotics ineffective in medicine (Assad and Signer 1990; Flavell et al., 1992; Nap et al. 1992; Fuchs et al., 1993; Daniell et al. 1998; Haldrup et al. 1998; Nielsen et al. 1998, Bertolla and Simonet 1999; Thimm et al. 2001; Dawla 2004). Flow of herbicide resistance marker genes into weeds can lead to emergence of ‘Superweeds’ (Eber et al. 1994; Frello et al. 1995; Bartsch and Pohl-Orf 1996; Lefol et al. 1996; Mikkelsen et al. 1996; Darmency et al. 1998; Bartsch and Ellstrand 1999; Daniell 1999; Wolfenbarger and Phifer 2000; Flothmann and van Aken 2001; Guadagnuolo et al. 2001; Coghlan, 2002; Dale et al. 2002; Auer 2003). In a nutshell, it can be presumed that the escape of antibiotic/herbicide marker genes from transgenics can lead to an ecological disturbance.
Growing concern about the use of marker genes that are ecologically unfit has forced researchers to look for alternate eco-friendly markers that can be safely used (Haldrup et al. 1998; Joersbo et al. 1998; Wang et al. 2000; Zhang et al. 2000; Zhang and Puonti-Kaerlas 2000; Lucca et al. 2001; Jaiwal et al. 2002; Puchta 2003; He et al. 2004). One of the approaches to identify eco-friendly markers focused attention on exploitation of certain essential biomolecules that can have a drastic negative effect on cellular metabolism and various developmental events, if present at levels above the threshold limit. Such biomolecules have been placed in the category of positive selection markers (Joersbo and Okkels 1996; Haldrup et al. 1998; Joersbo et al. 1999; Kaeppler et al. 2000; Negrotto et al. 2000; Wang et al. 2000, 2003; Joersbo 2001; Lucca et al. 2001; Wright et al. 2001; He et al. 2004).
Choline plays a vital role in several important cellular events such as (i) maintenance of structural and functional integrity of the membranes by regulating the levels of Ptd-Cho, often referred to as lecithin, and (ii) synthesis of an important compatible solute, glycinebetaine (GB) in plants (e.g. sugarbeet, spinach and barley), animals (e.g. rats) and microorganisms (e.g. E. coli). In general, choline has been demonstrated to play an important role in promoting plant growth, especially in the induction and growth of roots (Che et al. 1993). By regulating the production of glycinebetaine, choline even plays an important role in protecting cells against abiotic stresses such as salinity and drought (Summers and Weretilnyk 1993).
The perfect tuning in the levels of choline could be due to its toxicity to certain metabolic events whenever it is present in excess. Higher concentrations of choline have been shown to inhibit plant growth. For instance, high levels of choline inhibit activities of some enzymes such as Rubisco, glyceraldehyde-3-phosphate dehydrogenase, isocitrate dehydrogenase and malate dehydrogenase associated with some important metabolic events (Nash et al. 1982).
Choline is universally present in higher organisms (Prasad et al. 2000) and is primarily involved in maintaining structural and functional integrity of the membranes (Storey and Wyn Jones 1977). Living systems possess perfect mechanisms to regulate the level of free choline in their cells (Weretilnyk et al. 1995). In the presence of exogenous choline at levels as low as 50 μM, a marked decline in the specific activities of the enzymes involved in choline biosynthesis was reported by Mudd and Datko (1989a).
Based on the above-cited prior art there still remains a need for economically friendly selection markers which can be used in field surroundings and environments. Such selection markers must not bear the risk of escape from the plant, since unregulated and uncontrolled spread of markers in the environment is undesired.